Oxidative stress plays a critical role in the development of vascular disease (Baranano et al. (2002) “BILIVERDIN REDUCTASE: A MAJOR PHYSIOLOGIC CYTOPROTECTANT,” Proc. Natl. Acad. Sci. (USA) 99(25):16093-16098). Cells use multiple systems to protect against reactive oxygen species. The concentration of free heavy metals, which can catalyze the formation of free radicals, are tightly regulated by chelators such as ferritin and transferrin. Enzymes with antioxidant actions include catalase and superoxide dismutase, which together convert superoxide radicals into water. Small molecules, such as ascorbate and α-tocopherol act as direct antioxidants, quenching the propagation of free radicals. Glutathione occurs at millimolar concentrations in most tissues and is generally regarded as the principal endogenous intracellular small molecule antioxidant cytoprotectant.
Bilirubin is a lipophilic linear tetrapyrrole that occurs uniquely in mammals, and is abundant in plasma (Beri et al. (1993) “CHEMISTRY AND BIOLOGY OF HEME EFFECT OF METAL SALTS, ORGANOMETALS, AND METALLOPORPHYRINS ON HEME SYNTHESIS AND CATABOLISM, WITH SPECIAL REFERENCE TO CLINICAL IMPLICATIONS AND INTERACTIONS WITH CYTOCHROME P-450,” Drug Metab. Rev. 25(1-2): 49-152; Yamamoto (1968) “SYNTHESIS OF BILIRUBIN,” Naika Hokan. 15(11):391-398; Moore (1980) “THE BIOCHEMISTRY OF THE PORPHYRINS,” Clin. Haematol. 9(2):227-252). In humans, approximately 250-400 mg of bilirubin are formed daily as the final metabolic product of heme catabolism, as heme oxygenase (HO) cleaves the heme ring to form biliverdin (Yoshida et al. (2000) “MECHANISM OF HEME DEGRADATION BY HEME OXYGENASE,” J. Inorg. Biochem. 82(14):33-41; Montellano (2000) “THE MECHANISM OF HEMP OXYGENASE,” Curr. Opin. Chem. Biol. 4(2):221-227; Galbraith (1999) “HEME OXYGENASE: WHO NEEDS IT?,” Proc. Soc. Exp. Biol. Med. 222(3):299-305), which is then reduced by biliverdin reductase (BVR) to yield bilirubin (Wilks (2002) “HEME OXYGENASE EVOLUTION, STRUCTURE, AND MECHANISM,” Antioxid. Redox Signal. 4(4):603-614; Mantle (2002) “HAEM DEGRADATION IN ANIMALS AND PLANTS,” Biochem. Soc. Trans. 30(4):630-633; Ogawa (2002) “HEME METABOLISM IN STRESS RESPONSE,” Nippon Eiseigaku Zasshi 56(4):515-21). Approximately 80% of serum bilirubin is derived from hemoglobin of senescent erythrocytes that have been phagocytized by macrophages in the reticuloendothelial system (Shibahara et al. (2002) “HEME DEGRADATION AND HUMAN DISEASE: DIVERSITY IS THE SOUL OF LIFE,” Antioxid. Redox Signal. 4(4):593-602); the remainder derives from the catabolism of other haemoproteins and from the destruction of maturing red blood cells in the marrow.
Bilirubin has been found to possesses strong antioxidant potential against peroxyl and other reactive oxygen radicals (McGeary et al. (2003) “BIOLOGICAL PROPERTIES AND THERAPEUTIC POTENTIAL OF BILIRUBIN,” Mini Rev. Med. Chem. 3(3):253-256; Stocker et al. (1987) “BILIRUBIN IS AN ANTIOXIDANT OF POSSIBLE PHYSIOLOGICAL IMPORTANCE,” Science 235:1043-1046; Hidalgo et al. (1990) “CAN SERUM BILIRUBIN BE AN INDEX OF IN VIVO OXIDATIVE STRESS?” Med. Hypotheses 33(3):207-211; Stocker et al. (1987) “ANTIOXIDANT ACTIVITY OF ALBUMIN-BOUND BILIRUBIN,” Proc. Natl. Acad. Sci. USA 84:5918-5922; Machlin et al. (1987) “FREE RADICAL TISSUE DAMAGE: PROTECTIVE ROLE OF ANTIOXIDANT NUTRIENTS,” FASEB J. 1(6):441-445; Otterbein et al. (2003) “HEME OXYGENASE-1: UNLEASHING THE PROTECTIVE PROPERTIES OF HEME, Trends Immunol. 24(8):449-455; Wang et al. (2002) “BILIRUBIN AMELIORATES BLEOMYCIN-INDUCED PULMONARY FIBROSIS IN RATS,” Am. J. Respir Crit. Care Med. 165(3):406-411).
Several epidemiological studies have found that bilirubin levels are inversely associated with coronary artery disease and mortality from myocardial infarction (Scriver (1995) “THE METABOLIC AND MOLECULAR BASES OF INHERITED DISEASE,” McGraw-Hill, New York; Vitek et al. (2002) “GILBERT SYNDROME AND ISCHEMIC HEART DISEASE: A PROTECTIVE EFFECT OF ELEVATED BILIRUBIN LEVELS,” Atherosclerosis 160:449-456; Schwertner et al. (1994) “ASSOCIATION OF LOW SERUM CONCENTRATION OF BILIRUBIN WITH INCREASED RISK OF CORONARY ARTERY DISEASE,” Clin. Chem. 40:18-23; Hopkins et al. (1996) “HIGHER SERUM BILIRUBIN IS ASSOCIATED WITH DECREASED RISK FOR EARLY FAMILIAL CORONARY ARTERY DISEASE,” Arterioscler. Thromb. Vasc. Biol. 16:250-255; Djousse et al. (2001) “TOTAL SERUM BILIRUBIN AND RISK OF CARDIOVASCULAR DISEASE IN THE FRAMINGHAM OFFSPRING STUDY,” Am. J. Cardiol. 87:1196-200; Heyman et al. (1989) “RETINOPATHY OF PREMATURITY AND BILIRUBIN,” N. Engl. J. Med. 320:256; Temme et al. (2001) “SERUM BILIRUBIN AND 10-YEAR MORTALITY RISK IN A BELGIAN POPULATION,” Cancer Causes Control 12:887-894).
The possibility that the administration of bilirubin might find utility in providing cytoprotection is, however, encumbered by the toxicity and insolubility of the molecule (Hansen (2002) “MECHANISMS OF BILIRUBIN TOXICITY: CLINICAL IMPLICATIONS,” Clin. Perinatol. 29(4):765-778; Wennberg (1991) “CELLULAR BASIS OF BILIRUBIN TOXICITY,” NY State J. Med. 91(11):493-496; Bratlid (1991) “BILIRUBIN TOXICITY: PATHOPHYSIOLOGY AND ASSESSMENT OF RISK FACTORS,” NY State J. Med. 91(11):489-492). Excessive elevations of bilirubin lead to substantial deposits in the brain with the resultant kernicterus causing major brain damage (Baranano et al. (2002) “BILIVERDIN REDUCTASE: A MAJOR PHYSIOLOGIC CYTOPROTECTANT,” Proc. Natl. Acad. Sci. USA 99(25):16093-16098; Orth (1975) Virchows Arch. Pathol. Anat. 63:447-462).
In contrast to bilirubin, biliverdin is soluble. It can be produced by incubating bilirubin in the presence of a bilirubin oxidase (E.C.1.3.3.5). A number of enzymes with bilirubin oxidase activity from various plant sources are known (See U.S. Pat. No. 5,624,811; U.S. Pat. No. 4,985,360; U.S. Pat. No. 4,770,997; EP 0 140 004; EP 0 247 846; EP 0 005 637; EP 0 320 095; and DE 32 39 236).
Biliverdin has been proposed to be potentially useful as a cytoprotective therapeutic agent (Baranano et al. (2002) “BILIVERDIN REDUCTASE: A MAJOR PHYSIOLOGIC CYTOPROTECTANT,” Proc. Natl. Acad. Sci. USA 99(25):16093-16098; Colpaert et al. (2002) “INVESTIGATION OF THE POTENTIAL MODULATORY EFFECT OF BILIVERDIN, CARBON MONOXIDE AND BILIRUBIN ON NITRERGIC NEUROTRANSMISSION IN THE PIG GASTRIC FUNDUS,” Eur. J. Pharmacol. 457(2-3):177-86; Nakagami et al. (1992) “ANTIVIRAL ACTIVITY OF A BILE PIGMENT, BILIVERDIN, AGAINST HUMAN HERPESVIRUS 6 (HHV-6) IN VITRO,” Microbiol. Immunol. 36(4):381-390; Katori et al. (2002) “A NOVEL STRATEGY AGAINST ISCHEMIA AND REPERFUSION INJURY: CYTOPROTECTION WITH HEME OXYGENASE SYSTEM,” Transpl. Immunol. 9(2-4):227-233; Ryter et al. (2000) “THE HEME SYNTHESIS AND DEGRADATION PATHWAYS: ROLE IN OXIDANT SENSITIVITY. HEME OXYGENASE HAS BOTH PRO- AND ANTIOXIDANT PROPERTIES,” Free Radic. Biol. Med. 28(2):289-309; US 2003/0162826).
In particular, biliverdin has been proposed to be useful to treat vasoconstriction (US 2003/0027124), coronary artery disease (Vitek et al. (2002) “GILBERT SYNDROME AND ISCHEMIC HEART DISEASE: A PROTECTIVE EFFECT OF ELEVATED BILIRUBIN LEVELS,” Atherosclerosis 160:449-456; Schwertner et al. (1994) “ASSOCIATION OF LOW SERUM CONCENTRATION OF BILIRUBIN WITH INCREASED RISK OF CORONARY ARTERY DISEASE,” Clin. Chem. 40:18-23; Hopkins et al. (1996) “HIGHER SERUM BILIRUBIN IS ASSOCIATED WITH DECREASED RISK FOR EARLY FAMILIAL CORONARY ARTERY DISEASE,” Arterioscler. Thromb. Vase. Biol. 16:250-255; Djousse et al. (2001) “TOTAL SERUM BILIRUBIN AND RISK OF CARDIOVASCULAR DISEASE IN THE FRAMINGHAM OFFSPRING STUDY,” Am. J. Cardiol. 87:1196-200; Heyman et al. (1989) “RETINOPATHY OF PREMATURITY AND BILIRUBIN,” N. Engl. J. Med. 320:256; Temme et al. “SERUM BILIRUBIN AND 10-YEAR MORTALITY RISK IN A BELGIAN POPULATION,” (2001) Cancer Causes Control 12:887-894) and ischemia/reperfusion injury (Fondevila (2003) “BILIVERDIN PROTECTS RAT LIVERS FROM ISCHEMIA/REPERFUSION INJURY,” Transplant Proc. 35(5):1798-1799). Biliverdin has been found to block acetaminophen-induced injury (Chiu et al. (2002) “DIFFERENTIAL INDUCTION OF HEME OXYGENASE-1 IN MACROPHAGES AND HEPATOCYTES DURING ACETAMINOPHEN-INDUCED HEPATOTOXICITY IN THE RAT; EFFECTS OF HEMIN AND BILIVERDIN,” Toxicol. Appl. Pharmacol. 181(2):106-115), and liver graft injury (Kato et al. (2003) “BILIRUBIN RINSE: A SIMPLE PROTECTANT AGAINST THE RAT LIVER GRAFT INJURY MIMICKING HEME OXYGENASE-1 PRECONDITIONING,” Hepatology 38:364-373).
Despite improved methods of producing biliverdin, a need continues to exist for efficient and inexpensive methods for producing biliverdin, and in particular, methods capable of producing a single biliverdin isomer. The present invention is directed to such needs.